HCV is a significant human pathogen that has chronically infected 170 million people worldwide, including 4 million Americans. Clinical outcomes of HCV infections include cirrhosis and hepatocellular carcinoma. This year an estimated 10,000 individuals are expected to die from HCV infections in the U.S. and the number is expected to triple in the next 10 to 20 years. Although combination therapy with interferon (IFN) and ribavirin is effective in some patients, there is a high failure rate of about 50%. The lack of effective treatment for IFN-resistant patients coupled with severe adverse effects of the therapy, provides strong justification for development of alternative and effective therapeutic agents for HCV infection. 2-5A antisense is a potent and premising drug platform that can be adapted for HCV drug development. This strategy activates and recruits the ubiquitous antiviral enzyme, RNase L, to RNA targets. RNase L is activated by 2',5'-linked oligoadenylates (2-5A). By attaching a 2-5A cassette to antisense against sequences in HCV RNA, RNase L in intact cells is directed to specifically degrade HCV RNA. The goal of this proposal is to obtain a lead 2-5A anti-HCV drug that is active against a bread range of different HCV genotypes. Candidate drugs that target sequences in conserved regions of HCV RNA (present in 5' and 3' untranslated regions) will be screened and optimized to obtain the lead 2-5A anti-HCV compound. Specificity of the lead compound will be verified and its efficacy on different HCV genotypes will be determined. The success of the proposed studies will enable us to progress to drug safety and pharmacokinetics studies in the next phase and eventual marketing of a 2-5A anti-HCV drug for clinical applications.